Reactions between biological molecules exhibit an extremely high degree of specificity. It is this specificity that provides a living cell with the ability to carry out thousands of chemical reactions simultaneously in the same "vessel". In general, this specificity arises from the "fit" between two molecules having very complex surface topologies. For example, an antibody binds a molecule displaying an antigen on its surface because the antibody contains a pocket whose shape is the complement of a protruding area on the antigen. This type of specific binding between two molecules forms the basis of numerous biological assays.
For example, nucleic acids are linear polymers in which the linked monomers are chosen from a class of 4 possible sub-units. In addition to being capable of being linked together to form the polymers in question, each unit has a complementary sub-unit to which it can bind electrostatically. In the case of DNA, the polymers are constructed from four bases that are usually denoted by A, T, G, and C. The bases A and T are complementary to one another, and the bases G and C are complementary to one another. Consider two polymers that are aligned with one another. If the sequences in the polymers are such that an A in one chain is always matched to a T in the other chain and a C in one chain is always matched to a G in the other chain, then the two chains will be bound together by the electrostatic forces. Hence, an immobilized chain can be used to bind the complementary chain. This observation forms the basis of tests that detect the presence of DNA or RNA that is complementary to a known DNA or RNA chain. Such detection forms the basis of a number of medical and/or diagnostic tests.
The methods by which the binding of the mobile reactant to the immobilized component of the system is measured varies with the particular reactants. However, a significant fraction of all of the tests involve the measurement of a fluorescent dye that is associated with either the bound or mobile reactant. The dye may be attached to the reactant from the beginning of the process or it may be added through various chemical steps after the mobile and immobilized reactants have been brought into contact with one another.
The sensitivity of many assays is determined by the amount of non-specific binding that occurs between other macromolecules in the solution containing the mobile reactant and the substrate containing the bound reactant. The background material may bind to the substrate itself or to the bound reactant through binding reactions that are different from those for which the assay was designed.
The dye reactions utilized in prior art assay systems attach the dye molecule to a large class of macromolecules. For example, in antibody-antigen types of reactions in which the presence of antibody to a bound antigen is measured, the dye will be attached to any antibody that is bound to the substrate whether or not it is bound to the antigen. Similarly, in nucleic acid binding assays, the dye is bound to any double stranded region of nucleic acid. Hence, a mobile reactant that sticks to the surface of the assay plate and attracts its complementary strand is difficult to distinguish from a strand that is bound to the immobilized nucleic acid strand via the desired reaction. Similarly, a strand that binds by a partial sequence and is held in place by other binding reactions will also generate unwanted background.
Broadly, it is the object of the present invention to provide an improved binding assay.
It is a further object of the present invention to provide a binding assay that is less sensitive to non-specific binding than prior art binding reactions.
These and other objects of the present invention will become apparent to those skilled in the art from the following detailed description of the invention and the accompanying drawings.